Yuri Ozawa

Ca2+ Channel Subtypes and Pharmacology in the Kidney

A large body of evidence has accrued indicating that voltage-gated Ca(2+) channel subtypes, including L-, T-, N-, and P/Q-type, are present within renal vascular and tubular tissues, and the blockade of these Ca(2+) channels produces diverse actions on renal microcirculation. Because nifedipine acts exclusively on L-type Ca(2+) channels, the observation that nifedipine predominantly dilates afferent arterioles implicates intrarenal heterogeneity in the distribution of L-type Ca(2+) channels and suggests that it potentially causes glomerular hypertension. In contrast, recently developed Ca(2+) channel blockers (CCBs), including mibefradil and efonidipine, exert blocking action on L-type and T-type Ca(2+) channels and elicit vasodilation of afferent and efferent arterioles, which suggests the presence of T-type Ca(2+) channels in both arterioles and the distinct impact on intraglomerular pressure. Recently, aldosterone has been established as an aggravating factor in kidney disease, and T-type Ca(2+) channels mediate aldosterone release as well as its effect on renal efferent arteriolar tone. Furthermore, T-type CCBs are reported to exert inhibitory action on inflammatory process and renin secretion. Similarly, N-type Ca(2+) channels are present in nerve terminals, and the inhibition of neurotransmitter release by N-type CCBs (eg, cilnidipine) elicits dilation of afferent and efferent arterioles and reduces glomerular pressure. Collectively, the kidney is endowed with a variety of Ca(2+) channel subtypes, and the inhibition of these channels by their specific CCBs leads to variable impact on renal microcirculation. Furthermore, multifaceted activity of CCBs on T- and N-type Ca(2+) channels may offer additive benefits through nonhemodynamic mechanisms in the progression of chronic kidney disease.

Altered pressure-natriuresis in obese Zucker rats

It has not been examined whether the pressure-natriuresis response is altered in the insulin-resistant condition. Furthermore, despite an important role of nitric oxide (NO) in modulating pressure-natriuresis, no investigations have been conducted assessing the renal interstitial NO production in insulin resistance. The present study examined whether pressure-natriuresis was altered in insulin-resistant obese Zucker rats (OZ) and assessed the cortical and medullary nitrate/nitrite (NOx) levels with the use of the renal microdialysis technique. In OZ, serum insulin/glucose ratio (23.0+/-4.0x10(-8), n=9) and blood pressure (119+/-3 mm Hg) were greater than those in lean Zucker rats (LZ; 7.0+/-1.9x10(-8) and 103+/-4 mm Hg, n=9). The pressure-natriuresis curve in OZ was shifted to higher renal perfusion pressure (RPP), and the slope was blunted compared with that in LZ (0.073+/-0.015 vs 0.217+/-0.047 microEq/min kidney weight/mm Hg, P<0.05). The basal renal NOx level was reduced in OZ (cortex, 4.032+/-0.331 micromol/L; medulla, 4. 329+/-0.515 micromol/L) compared with that in LZ (cortex, 7.315+/-1. 102 micromol/L; medulla: 7.698+/-0.964 micromol/L). Furthermore, elevating RPP increased the medullary NOx in LZ, but this pressure-induced response was lost in OZ. Four-week treatment with troglitazone, an insulin-sensitizing agent, improved hyperinsulinemia, systemic hypertension, and basal renal NOx levels (cortex, 5.639+/-0.286 micromol/L; medulla, 5.978+/-0.284 micromol/L), and partially ameliorated the pressure-natriuresis curves; the slope of pressure-natriuresis curves and elevated RPP-induced NOx, however, were not corrected. In conclusion, our study suggests that insulin resistance is closely associated with abnormal pressure-natriuresis and hypertension. These deranged renal responses to insulin resistance are most likely attributed to impaired medullary NO production within the medulla.

Effect of T-Type Selective Calcium Antagonist on Renal Microcirculation: Studies in the Isolated Perfused Hydronephrotic Kidney

Although calcium antagonists exert preferential vasodilation of renal afferent arterioles, we have recently demonstrated that nilvadipine and efonidipine, possessing both L-type and T-type calcium channel blocking action, reverse the angiotensin (Ang) II–induced afferent and efferent arteriolar constriction. In the present study, we investigated the role of T-type calcium channels in mediating the Ang II–induced efferent arteriolar tone using the selective T-type calcium channel blocker mibefradil. Isolated perfused hydronephrotic rat kidneys were used for direct visualization of renal microcirculation. Administration of Ang II (0.3 nmol/L) caused marked constriction of afferent (from 13.5±0.6 to 9.2±0.6 &mgr;m, P <0.01, n=6) and efferent (from 11.5±1.0 to 7.4±0.7 &mgr;m, P <0.01, n=5) arterioles. Mibefradil (1 &mgr;mol/L) dilated both vessels, with 82±11% and 72±7% reversal of afferent and efferent arterioles, respectively. Similarly, nickel chloride (100 &mgr;mol/L) caused dilation of both arterioles, similar in magnitude in afferent (68±10%, n=7) and efferent (80±7%, n=7) arterioles. To eliminate the possibility that the mibefradil-induced dilation was mediated by L-type channel blockade, mibefradil was administered in the presence of nifedipine (1 &mgr;mol/L). Thus, nifedipine caused modest efferent arteriolar dilation (30±6% reversal, n=9), and subsequent addition of mibefradil elicited further dilation of this vessel (80±4%, P <0.01 versus nifedipine). Furthermore, mibefradil reversed the Ang II–induced efferent arteriolar constriction even in the presence of nifedipine and phentolamine. These findings demonstrate that T-type calcium antagonists markedly dilate the Ang II–induced efferent arteriolar constriction, but the action is not mediated by inhibition of catecholamine release. This potent activity would contribute to the efferent arteriolar response to nilvadipine and efonidipine and may offer benefit in light of glomerular hemodynamics.

Vessel- and vasoconstrictor-dependent role of Rho/Rho-kinase in renal microvascular tone

We examined the role of Rho/Rho-kinase in renal afferent and efferent arteriolar tone induced by angiotensin (Ang) II, KCl and elevated renal arterial pressure (from 80 to 180 mm Hg), using isolated perfused rat hydronephrotic kidney. In the condition with no vasoconstrictor stimuli, Y-27632, a Rho-kinase inhibitor, dilated only afferent (from 11.6 ± 0.4 to 14.1 ± 0.5 µm) but not efferent arterioles (from 11.6 ± 0.2 to 12.6 ± 0.7 µm) at 10–5 mol/l. During renal vasoconstriction by Ang II, Y-27632 restored the afferent arteriolar constriction (141 ± 10% reversal at 10–5 mol/l), whereas the ability of Y-27632 to inhibit the Ang II-induced efferent arteriolar constriction was diminished (73 ± 7% reversal). A similar action was observed with fasudil, another Rho-kinase inhibitor. Furthermore, Y-27632 impaired myogenic afferent arteriolar constriction, with 117 ± 17% inhibition at 10–5 mol/l. The inhibition by Y-27632 of the myogenic vasoconstriction was almost the same as that of the Ang II-induced tone of this vessel type. However, Y-27632 had a modest effect on KCl-induced vasoconstriction of afferent arterioles. In conclusion, the present study demonstrates a predominant role of Rho/Rho-kinase in mediating the basal and Ang II-induced tone of afferent, but not efferent, arterioles. Furthermore, the role of Rho/Rho-kinase in afferent arteriolar constriction differs, with a substantial contribution to Ang II-induced and myogenic constriction but a minimal role in depolarization-induced constriction. Since Ang II-induced, KCl-induced and myogenic constriction of afferent arterioles require calcium entry through voltage-dependent calcium channels, the interaction between Rho/Rho-kinase and the calcium entry pathway may determine the afferent arteriolar tone induced by these stimuli.

Differential Regulation of Elevated Renal Angiotensin II in Chronic Renal Ischemia

The present study was undertaken to clarify the role of intrarenal angiotensin (Ang) II and its generating pathways in clipped and nonclipped kidneys of 4-week unilateral renal artery stenosis in anesthetized dogs. After 4 weeks, renal plasma flow (RPF) decreased in clipped and nonclipped kidneys (baseline, 59±3; clipped, 16±1; nonclipped, 44±2 mL/min;P <0.01, n=22). Renal Ang I levels increased only in clipped, whereas intrarenal Ang II contents were elevated in both clipped (from 0.7±0.1 to 2.0±0.2 pg/mg tissue) and nonclipped kidneys (from 0.6±0.1 to 2.5±0.3 pg/mg tissue). Intrarenal ACE activity was increased in nonclipped kidneys but was unaltered in clipped kidneys. An angiotensin receptor antagonist (olmesartan medoxomil) given into the renal artery markedly restored RPF, and dilated both afferent and efferent arterioles (using intravital videomicroscopy). Furthermore, in clipped kidneys, the elevated Ang II was suppressed by a chymase inhibitor, chymostatin (from 2.1±0.6 to 0.8±0.1 pg/mg tissue;P <0.05), but not by cilazaprilat. In nonclipped kidneys, in contrast, cilazaprilat, but not chymostatin, potently inhibited the intrarenal Ang II generation (from 2.4±0.3 to 1.5±0.2 pg/mg tissue;P <0.05). Finally, [Pro11-d-Ala12]Ang I (an inactive precursor that yields Ang II by chymase but not by ACE; 1 to 50 nmol/kg) markedly elevated intrarenal Ang II in clipped, but not in nonclipped, kidneys. In conclusion, renal Ang II contents were elevated in both clipped and nonclipped kidneys, which contributed to the altered renal hemodynamics and microvascular tone. Furthermore, the mechanisms for intrarenal Ang II generation differ, and chymase activity is enhanced in clipped kidneys, whereas ACE-mediated Ang II generation is possibly responsible for elevated Ang II contents in nonclipped kidneys.

Free Radical Activity Depends on Underlying Vasoconstrictors in Renal Microcirculation

We examined the role of free radicals in renal microvascular tone induced by various vasoactive stimuli. Isolated perfused rat hydronephrotic kidneys were used for direct visualization of renal microcirculation. The effect of tempol on angiotensin II‐, norepinephrine‐, KCl‐, and pressure‐induced afferent arteriolar constriction was evaluated. Under angiotensin II‐induced constriction, tempol (3 mmol/L) caused 57 ± 8% dilation of afferent arterioles. In contrast, tempol elicited only 38 ± 8% and 26 ± 9% dilation of norepinephrine‐ and KCl‐induced constriction. Similarly, myogenic response induced by elevating renal arterial pressure from 80 to180 mmHg was resistant to the vasodilator action of tempol (22 ± 7% inhibition). Furthermore, tempol failed to reverse nitro‐L‐arginine methylester‐induced afferent constriction, nor had vasodilator effect on the angiotensin II‐induced constriction in the presence of nitro‐L‐arginine methylester. In contrast, nitroprusside elicited marked vasodilation of angiotensin II‐ (97 ± 5% reversal) and norepinephrine‐induced afferent constriction (89 ± 6% reversal), but had less effect on KCl‐ (46 ± 8% reversal) and pressure‐induced constriction (26 ± 9% reversal). These different actions were also observed when polyethylene‐glycolated superoxide dismutase was used as an antioxidant. In conclusion, the role of free radicals in afferent arteriolar tone varies, depending on the underlying vasoconstrictor stimuli, with greater contribution of free radicals to angiotensin II‐induced constriction. The heterogeneity in the responsiveness to free radical scavengers is attributed to both magnitude of free radicals produced and sensitivity of the underlying vasoconstrictors to nitric oxide.

Impaired nitric oxide- and endothelium-derived hyperpolarizing factor-dependent dilation of renal afferent arteriole in Dahl salt-sensitive rats.

Background and Aims:u2003 We previously demonstrated that acetylcholine elicited nitric oxide‐dependent sustained and endothelium‐derived hyperpolarizing factor (EDHF)‐dependent transient dilation of afferent arterioles. The present study examined whether free radicals interacted with nitric oxide‐dependent and EDHF‐dependent vasodilator mechanisms in renal microvessels of salt‐sensitive hypertension, using the isolated perfused hydronephrotic kidney.

Role of nitric oxide and prostaglandin E2 in acute renal hypoperfusion

SUMMARY: Although acute renal ischaemia alters the production of various paracrines, there has been little investigation examining the role of intrarenal vasoactive substances. In the present study, we investigated the role of intrarenal nitric oxide and prostaglandins in modulating the acute renal hypoperfusion‐induced alterations in renal function. After a 90% clipping of the left renal artery for 60u2003min, the clip was released, and the renal haemodynamics and sodium excretion were evaluated in both clipped and non‐clipped kidneys of anaesthetized dogs. Furthermore, the changes in renal contents of nitrate/nitrite (NOx) and prostaglandin E2 (PGE2) were assessed by using the renal microdialysis technique. The release of the clipping elicited a gradual recovery of renal plasma flow and glomerular filtration rate, and a sustained increase in fractional sodium excretion (FENa) in the clipped kidney. Renal interstitial NOx was reduced in both the cortex (from 8.2u2003±u20031.1 to 2.5u2003±u20030.3u2003µmol/L, Pu2003<u20030.01) and medulla (from 10.1u2003±u20030.9 to 3.1u2003±u20030.2u2003µmol/L, Pu2003<u20030.01), but the levels gradually elevated after declamping. The treatment with nitro‐l‐arginine methylester only modestly impaired the recovery of renal plasma flow (RPF; at hour 4) and glomerular filtration rate (GFR; at hours 3 and 4 after declamping), without affecting FENa. Conversely, the renal PGE2 levels increased prominently upon the onset of ischaemia (medulla, from 149u2003±u200319 to 378u2003±u200339u2003pg/mL, Pu2003<u20030.01; cortex, from 107u2003±u200313 to 302u2003±u200334u2003pg/mL, Pu2003<u20030.01). Furthermore, the pretreatment with a non‐specific cyclo‐oxygenase (COX) inhibitor, sulpyrine, and a COX‐2‐specific inhibitor, NS398, prominently inhibited the increases in FENa induced by the acute renal arterial clipping in a similar manner. In conclusion, in acute renal hypoperfusion, nitric oxide (NO) plays a permissive role in the recovery of the renal haemodynamics. In contrast, sustained increases in renal PGE2 in both clipped and non‐clipped kidneys indicate that the COX‐2‐mediated PGE2 contributes importantly to the failure of the sodium reabsorption in response to acute renal hypoperfusion.

Divergent natriuretic action of calcium channel antagonists in mongrel dogs : renal haemodynamics as a determinant of natriuresis

This study examined the effects of different types of calcium channel antagonists on renal haemodynamics and natriuresis. The intravenous infusion of nifedipine (L-type blocker), efonidipine (L/T-type blocker) or mibefradil (predominant T-type blocker) into anaesthetized dogs elicited similar, albeit modest, reductions in blood pressure. Nifedipine (1 microgram.min(-1).kg(-1)) increased renal plasma flow (RPF) (23+/-6%; P<0.05) and glomerular filtration rate (GFR) (25+/-5%; P<0.05) (all values are means+/-S.E.M., n=7). Efonidipine (0.33 microgram .min(-1).kg(-1)) also elevated RPF (18+/-6%; P<0.05), and tended to increase GFR (17+/-8%; P=0.08). These antagonists exerted contrasting actions on the filtration fraction (FF), with an increase being elicited by nifedipine, whereas efonidipine had no effect. Furthermore, mibefradil (0.01-1 microgram.min(-1).kg(-1)) slightly elevated RPF (between 5+/-3% and 8+/-3%), but failed to alter GFR, resulting in a decrease in FF. Nifedipine slightly increased urinary sodium excretion (U(Na)V) (29+/-16% increase at 1 microgram .min(-1).kg(-1)) and fractional sodium excretion (FE(Na)) (18+/-14%), whereas efonidipine (0.33 microgram .min(-1).kg(-1)) elicited marked elevations in U(Na)V (110+/-38%; P<0.05) and FE(Na) (102+/-44%; P<0.05). Mibefradil (1 microgram .min(-1).kg(-1)) exerted a moderate natriuretic action [U(Na)V, +60+/-32% (P=0.1); FE(Na), +67+/-20% (P<0.05)]. Furthermore, although a positive correlation was observed between U(Na)V and urinary nitrate/nitrite excretion, no differences were noted between the various calcium channel antagonists. Collectively, this study demonstrates that the glomerular haemodynamic and natriuretic actions of these calcium channel antagonists, which possess diverse blocking activities on L/T-type channels, vary. Based on the divergent actions on FF (i.e. increase, no change and decrease by nifedipine, efonidipine and mibefradil respectively), the natriuretic action of calcium channel antagonists is possibly attributed to the inhibition of tubular sodium reabsorption associated with increased post-glomerular blood flow, rather than increased GFR.

Distinct role of nitric oxide and endothelium-derived hyperpolarizing factor in renal microcirculation. Studies in the isolated perfused hydronephrotic kidney.

Background: Both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) are established as important factors determining the vascular tone. The relative contribution of these factors to the renal microvascular tone, however, has not been delineated. Methods: Isolated perfused hydronephrotic rat kidneys were used to characterize the relative role of NO and EDHF in mediating the tone of interlobular arteries (ILA) and afferent arterioles (AFF). Results: During the norepinephrine constriction, acetylcholine (ACH, 1 µmol/l) induced a sustained vasodilation of ILA (90 ± 9% reversal) and AFF (117 ± 13% reversal). In the presence of nitro-L-arginine methylester (LNAME), the ACH-induced vasodilation of ILA and AFF was converted to transient dilation, with only 53 ± 7 and 32 ± 7% reversal observed 10 min after 1 µmol/l ACH (i.e sustained phase). In contrast, LNAME had no effect on the initial phase of ACH-induced dilation. In the presence of apamin + charybdotoxin, the initial vasodilator response to ACH (1 µmol/l) was diminished (ILA, from 108 ± 8 to 46 ± 9%; AFF, from 108 ± 14 to 58 ± 8%), whereas no impairment was observed in sustained phases. Furthermore, the magnitude of the vasoconstriction caused by LNAME was greater at smaller vessel segments. Finally, the LNAME-induced inhibition of the sustained phase of ACH-induced vasodilation was greater as the vessel diameter decreased. Conclusions: That the relative contribution of NO and EDHF differs, with a greater role of NO in the basal tone and ACH-induced vasodilation at smaller vascular segments of ILA and AFF.